ABSTRACT: Stroke is a leading cause of death and disability and the risk for stroke increases with age. The mechanisms that contribute to post-ischemic injury and neurological dysfunction after stroke are not completely understood, and most clinical trials conducted to date for stroke therapeutics have failed. Failure to consider white matter (WM) injury is a critical gap in the development of successful stroke therapy. As mechanisms of WM injury differ from those in gray matter and change with age, an ideal stroke therapeutic must not only be directed towards neuronal and axonal protection across age, but also must restore function when applied after injury. One of our significant research achievements during the previous funding cycle established that Class I histone deacetylase (HDAC) activation contributes to excitotoxicity during ischemia and contributes to oxidative injury during the post-ischemic period by impairing mitochondrial structure and function. Class I HDAC inhibition promotes axon function recovery when applied before or after ischemia in young and aging WM through unknown mechanisms. Our current proposal focuses on the mechanisms of post-ischemic protection conferred by Class I HDAC inhibition in young and aging WM. Post-ischemic benefits of HDAC inhibition correlated with mitochondrial integrity, ATP production, and promotion of axon function recovery. While little is known about the gene regulatory mechanisms underlying this protective phenomenon, an intriguing reciprocal relationship has emerged between levels of HDACs and miRNAs affecting cellular survival following stroke. Among ischemia-regulated miRNAs, miR-331 is predicted to target Class I HDACs. Ischemia up-regulates Class I HDAC levels in young and aging WM. Our preliminary findings show that ischemia led to decreased levels of miR-331 concomitant with increased HDAC expression and that HDAC inhibition up-regulated miR-331 above control levels. Furthermore, WM ischemia activates nitric oxide synthase (NOS), leading to oxidative injury via mitochondrial dysfunction. In light of this information, we propose to further extend these studies by testing our novel hypothesis that Class I HDAC activation mediates WM ischemic injury by contributing to increased oxidative stress, impairing mitochondrial function, and down-regulating glial expression of miR-331. Our overall goal is to determine whether Class I HDACs act directly or recruit NOS or interact with miR-331 to exert post-ischemic injury to young and aging WM. Electrophysiological, biochemical, and mouse genetic approaches will be employed to test the following Specific Aims: Aim 1 is designed to investigate how the cellular origins of NOS isoform activation change with age and ischemia; Aim 2 is designed to determine whether Class I HDAC activation recruits NOS or directly mediates mitochondrial injury during ischemia; and Aim 3 is designed to establish whether Class I HDACs interact with miR-331 to mediate ischemic WM injury. Overall, the present project will unravel the role of Class I HDAC activity in WM ischemic injury to design novel therapies to minimize post-ischemic injury in patients.